“Experimental and Evidence based Protocols in Pharmacology” provides a holistic coverage of scientific protocols involved in the pharmacology and toxicological studies. The book serves as a reference to the biomedical researchers working in pharmacology and allied subjects to mitigate the challenges in the routine laboratory procedures. Book has 22 chapters with evidence based protocols and procedures to conduct the defined experiment. Further, this book provides single reference window with evidence based practical approaches to cater the need of academician and research scholars. The book is designed in a diligent manner to provide majority of protocols viz. Primary cell culture, Mesenchymal stem cells isolation and its application, DNA fragmentation Assay, oxidative stress evaluation protocols, Cytotoxicity and Genotoxicity Assays, Apoptosis studies, Analytical studies involving HPLC, GC-MS, ICP-OES and experimental pharmacological tool in smooth muscle pharmacological studies etc. to cement the scientific acumen. This book imbibes all the basic and advanced research protocols along with their translational approaches in experiments involved in pharmacology and also provides the insight of example based drug assays to help in bioprospecting of naive molecules. Further, this book also provide a reference book to the researchers involved in toxicological testing using OECD testing guideline to calculate the lethal dose of any compound in a better manner. This MS also suffice the different methods to calculate the median lethal dose (LD50 ). The important reproductive toxicity assays to ascertain the toxic potential of any xenobiotics is also incorporated in this MS. Moreover, sophisticated technique like Flow cytometry and other experimental models viz. Direct and Indirect LPS induced acute lung injury model has also well written to provide an insight of the experimentation involving laboratory animals.

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed. Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials. Cytotoxicity Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison. In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death). Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome. Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic. Why we Need to Evaluate Genotoxicity? Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

Apoptosis is triggered by extrinsic and intrinsic stimuli as radiation, oxidative stress and genotoxic chemicals. It is defined by characteristic changes in the nucleus morphology including chromatin condensation and fragmentation, overall cell shrinkage/rounding and formation of apoptotic cell bodies. Among the many markers of apoptosis, DNA fragmentation is considered a hallmark. The DNA ladder assay described here is a simple, sensitive, cost-effective and rapid method for estimating apoptosis in cells. Isolation of Genomic DNA Genomic DNA from primary cells culture of PMNC and splenocytes of rats is isolated by using standard phenol-chloroform DNA isolation protocol of Sambrook and Russel (2001) as described below: 1. Preparation of primary cells culture of different cells, namely-PBMNCs, thymocytes and splenocytes form Wistar rats and viability checked by trypan blue dye exclusion test. 2. 106 cells/ml of primary cell culture is used and seeded in 24 well culture plates containing along with treatment with xenobiotics at different µM concentration. 3. Cell culture plates are incubated in CO2 incubator at 37°C with 5% CO2 for 12 hrs. 4. The cells are collected in 1.5 ml of micro centrifuge tube and centrifuged at 3200 rpm for 10 min at 4°C; supernatant is discarded and pellet is washed with PBS (pH 7.2). 5. DNA extraction buffer (500 µl/tube) is added in tubes. Gently tap to disperse the cell pellet in extraction buffer and keep in water bath for 1.0 hr at 37 °C (it is to ensure that pellet is completely suspended so that cells are accessible to SDS and Proteinase K). 6. 10 % SDS is added (20 µl/ml) to cell suspension and the tubes are gently mixed by inverting the tube. The contents of tube become viscous which indicates lysis of cells. After that sample is handled gently from onward to avoid shearing of DNA. 7. Proteinase K (15 µl) of 20 mg Proteinase K/ml of buffer is added in tubes in two pulse i.e. half the requirement is added to tube in 1st pulse, mixed gently end to end and kept in water bath at 50°C. After 3-4 hrs, the second pulse of the remaining amount of Proteinase K is added and tubes are incubated at 50°C overnight. 8. Equal volume of equilibrated phenol (Tris saturated phenol pH> 7.8 ) is added in the tubes next day morning. Mix the contents of tube gently by inverting the tube for 15 min till a light coffee coloured uniform solution without any balls of phenol is formed and then centrifuge at 3400 rpm for 15 min.

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell. Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008). Experimental Procedure Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows: 1. Preparation of primary cells culture as described in earlier methods. 2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method. 3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent. 4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes. 5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Spontaneous contractions of the uterus are controlled and coordinated for various reproductive functions. The non-pregnant uterus facilitates ova and sperm transport, provides adequate embryo placement for implantation, and contributes to the expulsion of menstrual debris through increased contractility, or periods of relative quiescence (Hutchings et al., 2009). Uterine dysmotility has been implicated in the pathogenesis of certain infertilities, implantation failure, endometriosis, dysmenorrhea, and abnormal menstrual events (Kunz and Leyendecker, 2002; Aguilar and Mitchell, 2010). Although these conditions may not contribute to mortality but can significantly affect patient’s reproductive health, and considerably alter their quality of life (Jones et al., 2004; Weissman et al., 2004; Chachamovich et al., 2010). Dysmenorrhoea refers to the occurrence of painful cramps in the lower abdominal region during menstruation and is one of the unresolved gynecological disorders in adolescent girls and women of reproductive age (Wang et al., 2004; Harel, 2008). This condition is mainly characterized by cramping pain in the lower abdomen immediately before or during menstruation, which does affect the quality of their life and work (Davis and Westhoff, 2001).

Introduction Acute lung injury (ALI) is the manifestation of an inflammatory response of lung resulting from severe sepsis, bacterial or viral pneumonia, trauma, and burns in animals (Ali et al., 2020).It is distinguwashed by disruption to the alveolar capillary membranes, atelectasis of the pulmonary airspaces, and neutrophil and protein-rich fluid infiltration into the alveolar space (Li et al., 2019). Although advances in treatment, the disability and fatality rates of ALI remain high, that is around 30-40% (Ali et al., 2020). At the moment, new therapeutic alternatives are urgently required to treat ALI. As a result, this is a very active area of research to understand the molecular principles underlying pulmonary inflammation and ALI (Reiss et al., 2012). The use of animal models of inflammatory lung has allowed for the probing of pathways of inflammation responsible for injury. Specially, rodent models may serve a link between in vitro research to large animals and clinical studies by allowing for high quantities of samples under physiological circumstances at low expenses (Domscheit et al., 2020).LPS, found in the outer membrane of gram-negative bacteria, consists of a polar lipid head group (lipid A) and a chain of repeating disaccharides (Gao et al., 2023). LPS induced lung injury model, closely mimic sepsis and inflammatory lung diseases, is a well-establwashed model in this field of research (Reiss et al., 2012). To mimic acute lung injury, LPS can be administered either directly through intratracheal or intranasal route (direct pulmonary insult) or indirectly through intraperitoneally and intravenous routes (indirect extra pulmonary insult) (Perl et al., 2011). This chapter focuses mostly on rodent model of acute lung injury caused by lipopolysaccharide (LPS), both direct and indirect. Additionally, it also includes the characteristics and underlying molecular mechanism of LPS induced lung injury along with applications in drug development.

Mesenchymal Stem Cells Recent advances in the field of regenerative medicine have opened up newer therapeutic modalities using cell-based and cell-free therapies, particularly stem cells, as potential candidates. As per the definition, stem cells are the undifferentiated progenitor cells that can regenerate and can also differentiate into various specialized cells (Hoang et al., 2022). They can be classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and postnatal adult stem cells (Locke et al., 2009). Among postnatal adult stem cells, mesenchymal stem cells (MSCs) have emerged as a popular choice of candidate for therapeutics due to the relatively easy availability, simple isolation techniques, low risk of teratogenicity, minimal ethical and legal concerns unlike embryonic stem cells, hence, are easy to apply in clinical research and practice (Voga et al., 2020). Among MSCs of different origin, bone marrow and particularly adipose tissue-derived stem cells (ADMSCs) received more attention and are one of the most common and promising stem cell types. These cells can be simply obtained from subcutaneous and omental fat or liposuction aspirates for the treatment of diverse clinical applications (Kim &Heo, 2014). These cells can also be expanded vigorously in vitro and differentiated into specific cell lineages such as adipocytes, osteoblasts, chondrocytes, and neurocytes and are even genetically stable in long-term cultures (Miana& González, 2018). Mesenchymal stem cells (MSCs) are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the (ISCT) International Society for Cell and Gene Therapy (Dominici et al., 2006).

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Flow Cytometry is a tool used to detect and measure chemicophysical characteristics of a population of cellular particles. Modern day biomedical research needs the molecular assembly of tools to proceed in the advent of biomedical research with real time diagnostic, prognostic and experimental detection of markers. For the same, a naïve tool is flow cytometry. In this technique, a sample having cells or particles is suspended into a fluid and injected into instrument. The ability to perform these measurements in a very rapid time span is one of the key advantages of the flow cytometry process. A single-cell suspension must first be prepared in order to analyse solid Tissues. A flow cytometer is look alike to microscope, except that, instead of producing an image of the cell, it generally offers a cell-by-cell basis high-throughput, automated quantification of specified optical parameters. They can quantify up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute. Sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. These cells are tagged with fluorescent markers in order to absorb the light and then emitted them in the form of band of wavelengths. Average ten thousand cells can be quickly examined and the data gathered are processed by a computer.

High-performance liquid chromatography or high-pressure liquid chromato graphy (HPLC) is a chromatographic method that is used to separate a mixture of compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the individual components of the mixture. It involves the injection of a small volume of liquid sample into a tube packed with tiny particles (1.8- to 5-micron (μm) particle size called the stationary phase) where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”. Aflatoxins are toxic and carcinogenic compounds produced as metabolites by many Aspergillus species. This fungus invades habitats such as soil, grain, nuts, or decaying vegetation when- ever suitable conditions such as high moisture and temperature are met. B1 is the most toxic of at least 14 different types of naturally occurring aflatoxins. Therefore, many countries and regulatory agencies impose strict limits on B1 as well as other common aflatoxins such as B2, G1, and G2 despite exhibiting lower toxicity. The U.S. FDA set the action level to20 μg/kg (ppb) for human food and to 300 μg/kg (ppb) for animal feed. Aflatoxin B1 is extremely widespread and is present in food and feed products such as cottonseed, corn, and peanuts. It is highly toxic and has been classified as a group 1 carcinogen by the WHO. The aflatoxins B2 , G1 , and G2 are usually found accompanying B1 , in lower concentrations in the contaminated samples. The order of toxicity is AFB1 > AFG1 > AFB2> AFG2.

Introduction Minerals are present in all body tissues and fluids to carry out the physicochemical processes of life; both in animals and human. It is broadly classified into two categories: (1) macro and (ii) micro or trace elements. The macro elements include calcium, phosphorus, potassium, magnesium, sulfur, sodium and chloride, while elements such as iron, copper, cobalt, iodine, zinc, manganese, molybdenum, selenium and chromium come under micro elements. Animals need large amounts of calcium for construction and maintenance of bone and normal function of nerves and muscles. Phosphorus is a vital constituent of adenosine triphosphate (ATP) and nucleic acid and is also essential for acid-base balance, bone and tooth formation. Iron is an important component of the cytochromes for carrying out cellular respiration in addition to its vital role in red blood cells. Magnesium, copper, selenium, zinc, iron, manganese and molybdenum are important co-factors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways. Animals need iodine to make thyroid hormones. Sodium, potassium and chlorine are important in the maintenance of osmotic balance between cells and the interstitial fluid. Excessive intake of some minerals can upset homeostatic balance and cause toxic side effects.

Isolated tissue preparations are widely used in physiological or pharmaco logical research to delineate the mechanism of action of drugs or any active biomolecules. Traditionally it is used as a tool for bioassay of drugs. The tissue used for such pharmacodynamics studies may be isolated from different animal models viz. frog, rodents, rabbits, sheep, goat, buffalo, chicken etc. Evaluation of drug-receptor interaction and underlying mechanism of action is then performed using isolated organ bath or myograph where the isolated tissues are mounted under optimum resting tension and changes in the tension following exposure of drug is recorded with the help of recording device. With the advancement of science and technology, the recording devices are also gradually upgraded from smoked drum to student’s physiograph to the most advanced Data Acquisition System (DAS) based polyphysiograph. The isolated tissue preparations (uterine strips, arterial rings, tracheal chains etc.) need essential and appropriate nutrients and environment during experimental which is generally provided by using suitable salt solutions (Physiological Salt Solutions) which provide ionic balance, energy supply, maintain pH and tissue tonicity. The composition of physiological salt solutions is such that it provides an artificial media simulating with the inorganic composition of blood plasma together with a buffer mechanism to maintain an optimum pH of 7.0 to 7.4 and glucose to facilitate tissue metabolism. Commonly used PSS are Frog ringers, tyrode, Kreb’s, de Jalon, Ringer-Locke, modified Kreb’s Henseleit solution etc.

The cytological insult due to exposure of different environmental pollutant viz. pesticides, herbicides etc. impose great loss to the vitality and health of the individuals. The basic pathophysiological distrubances following the expo sure of these deterrents to the different visceral organs leads to dysfunction of these organs. Imbalance between body’s antioxidant defense and peroxidation of the lipid layer of cells’ causing free radical generation is defined as Oxidative stress. Free radicals are the molecules or entity that contains one or more than one unpaired electron. Pathophysiology of several diseases are associated with the generation of oxidative insult and many xenobiotics associated toxicity are also a result of oxidative damage and generation of reactive oxygen species and reactive nitrogen species. Alzheimer’s Disease is one of the fine example of oxidative damage associated neuronal degeneration. Oxidative stress can be estimated using following parameters: 1. Lipid peroxidation (Paula et al., 2005) 2. Reduced glutathione (Sedlak and Lindsay, 1968) 3. Superoxide dismutase (Madesh and Balasubramanian, 1998) 4. Catalase (Aebi, 1983)

Tissue Collection Rats are most commonly employed as test subjects to conduct the experiments related to drug metabolizing enzyme assays involving various enzymes i.e. microsomal and non microsomal enzymes. Rats are humanely sacrificed at the end of the exposure period of 28 days of any therapeutic agents or drug and the liver of all the animals has to excise and immediately wash with ice-cold normal saline, weigh and perfuse with ice-cold 1.15% KCl solution containing 0.05 mM EDTA. Livers are processed further for preparation of microsomes for determination of microsomal drug metabolizing enzymes. Preparation of Microsomes Livers tissues are homogenized in 3 volumes (1:3) of ice-cold (1-4o C) homogenization buffer containing 1.15% potassium chloride in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates are centrifuged at 15000 g for 30 min (Hitachi cs120GX II). The mitochondrial supernatant (8 ml) is transferred to ultracentrifuge tubes and spun at 105000 g for 1 hour in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions are collected from the middle separating the lipid layer on top in separate micro centrifuge tubes. The microsomal pellet is then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet so obtained is resuspended in 4 ml of resuspension buffer and 1 ml aliquots are stored immediately at -80o C until assayed for drug metabolizing enzymes.

Acetylcholinesterase activity in brain is measured by the method described by Ellman’s et al. (1961). Reagents 1. Phosphate buffer (0.1M) (pH 8.0) Solution A: 5.22 g of K2 HPO4 and 4.68 g of NaH2 PO4 are dissolved in 150 ml of distilled water. Solution B: 6.2 g of NaOH is dissolved in 150 ml of distilled water. Solution B is added to solution A to get desire pH 8.0 and then finally the volume is made up to 300 ml with distilled water. 2. DTNB reagent 39.6 mg of DTNB with 15 mg of NaHCO3 is dissolved in 10 ml of 0.1 M phosphate buffer. 3. Acetylthiocholine (ATC) 21.67 mg of Acetylthiocholine is dissolved in 1 ml of distilled water. Procedure The brain tissue is weighed and 10% homogenate is made in 0.1M Phosphate buffer (pH 8.0). Take 0.4 ml aliquot of homogenate in a cuvette containing 2.6 ml phosphate buffer (0.1M) and 100 µl DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reached a stable value, it is recorded as the basal reading .To this 20 µl of acetylthiocholine is added and change in absorbance is recorded for a period of 10 min at intervals of 2 min. Change in the absorbance per minute determined.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using a large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of the drug discovery process can save time and money both by eliminating least beneficial NCEs. Preparation of Reagents Culture media For the culture of hepatocytes medium M199 (GibcoBRL, USA) is used. One pouch of the dry media is dissolved in 1 liter of filtered distil water. The pH of the media is adjusted by adding 0.4% sodium bicarbonate to be around 7.4. The media is than sterilized by filtration through seitz filter (0.22 mm) and stored in 50 ml sterilized conical flasks.

Isolation of Peripheral Blood Lymphocytes Chemicals 1. Heparin 2. Sterile PBS (pH 7.2) 3. Histopaque-1077 4. RPMI-1640 Media Protocol Collect 5 ml of blood from the animal in 1 ml (20 IU/ml of blood) of diluted heparin and add 2 ml of PBS. Layer over the diluted blood on 5 ml of histopaque-1077 in 15 ml centrifuge tube and centrifuge at 1500 rpm for 40 min. Collect the buffy coat at the interface of plasma and histopaque in separate centrifuge tubes. Wash the cells thrice with PBS and suspended in 2 ml of RPMI-1640 media supplemented with 10% fetal calf serum. Determine the percentage cell viability by 0.1% trypan blue exclusion test, using hemocytometer. Finally dilute the cell suspension to 106 cells/ml in RPMI 1640 medium.

LD 50- It is defined as the concentration/dose that kills 50 % of the experimental population. Acute toxic studies have been conducted in order to calculate LD50. There are several methods to calculate LD50, some are as follows: Methods to Determine LD 50 • Karber’s method. • Miller & Tainter method • Lorke method • Fixed dose method • Acute Toxic Class method • Up & down procedure Karber’s Method This method involves the administration of different doses of test substance to various groups, which has five animals each. The first group of animals is administered with the vehicle in which the test substance was dissolved or diluted

Introduction The immense use of chemicals and pharmacological entities has put the public health at brisk of potential health hazards. These substances in modern days are presented in the form of common constituents of food, medicines, beverages and junk foods etc. However, long term consumption of these substances might be responsible for chronic toxicity, but the accidental ingestion of large quantities of these substances may result into adverse acute toxic reactions. Hence an experimenter should have the acumen to develop and perform the acute toxic tests in order to find out the lethal dose 50 i.e. LD50 of the substances. Acute Toxicity Studies Acute toxicity studies helps to establish the dose dependent adverse effect including mortalities. The LD50 has been used as a major parameter in screening of pharmacological agents for acute toxic potential. Apart from mortality, other biological effects and the time of onset, duration and degree of recovery on survived animals, are also important in evaluation of acute toxicity. Hence, acute toxicity study solely gives information about LD50, therapeutic index and the degree of safety of a pharmacological agent. Acute toxicity is defined as adverse effects occur following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. Oral administration is the most common form of acute systemic toxicity testing.

Epididymal Total Sperm Count The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003). Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor) Liveability and abnormal sperm Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa. The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain. • A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide. • 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop. • Epididymal semen and stain are mixed gently using a blunt fine glass rod. • After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide. • The smear is examined under oil immersion objective.

A Acetylcholinesterase activity 105 Acute lung injury (ALI) 41, 42, 44, 45 Aflatoxins 73, 74, 75 Aflatoxins B1 74 Analyzing environmental samples 65 Apoptosis 7, 15, 25, 27, 29, 115, 116